Proteomics is a newly emerging field in the post-genomics era1. A major activity of proteomics is the identification of unique proteins in cellular complexes in a high throughput mode2. Peptide mass mapping followed by database searching is a major approach towards the identification of a protein using mass spectrometry (MS). Using this approach the measured and calculated masses of proteolytic peptides are compared for a best mass-fit to possible proteins3,4. The most commonly used method is an in-gel digestion of the protein spots separated by two dimensional polyacrylamide gel electrophoresis (2D PAGE) for analysis by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS5,6. Mass accuracy and precision are of prime importance to ensure specificity of the search for a target protein in database searches.
The mass-to-charge (m/z) ratios of a large number of proteolytic peptides covering much of the protein sequence must be precisely determined. Too few proteolytic peptides from a target protein in a MALDI-TOF MS spectrum reduces the specificity and precision of the database search and can give false positives. Currently, the typical sequence coverage of a protein in a MALDI-TOF MS spectrum is less than 40%7-14. This depends largely on sample availability7, sample preparation methods8, matrix solution conditions9, and matrix crystal morphology10, as well as the physical properties of proteins such as charged side chains11,12, peptide hydrophobicity13, and the potential to form stable secondary structures14. In most cases, MS data acquisition and interpretation have proven to be time-consuming in the identification of unique proteins in complexes because of problems such as low sample availability, background or artifact ions, mass degeneracy of peptides from protein impurities and post-synthetic modifications of proteins as examples15 Ultrahigh mass accuracy provided by high-cost instruments is often required to determine the absolute m/z values of these proteolytic fragments16,17. To increase the specificity of identification of proteolytic peptides, the external labeling of the C-termini of tryptic peptides with H2O containing 50% 18O during trypsin digestion has been used18,19. Although this is a useful method for excluding unrelated peaks from the data search, its selectivity and sensitivity is poor because only the C-termini of all tryptic peptides are labeled with 18O.
It is necessary to extend the limited resource of peptide signals available in MALDI-TOF MS spectra for characterizing proteins by further increasing the specificity of proteolytic peptide identification. Stable isotope labeling; that is, the replacement of 13C for 12C, 15N for 14N, or 2H for 1H, in proteins or DNA oligomers can generate internal mass “signatures” with characteristic mass shifts in their isotopic distribution patterns without affecting their chemical and structural properties20. Uniformly 15N-labeled proteins have been generated for the accurate MS-based quantitation of protein expression21 and for improvements in the sensitivity and accuracy of molecular mass measurements22.
Stable isotope 13C/15N-labeled nucleotides have successfully been incorporated as internal markers to determine the nucleotide composition of PCR products23.
Accordingly, it is an object of the present invention to increase the specificity of mass spectrometric proteolytic peptide identification.
Additional objects, advantages and novel features of the invention will be set forth in part in the description which follows, and in part will become apparent to those skilled in the art upon examination of the following or may be learned by practice of the invention. The objects and advantages of the invention may be realized and attained by means of the instrumentalities and combinations particularly pointed out in the appended claims.